Despite recent advances in the medical management of pulmonary arterial hypertension (PAH), survival rates in the U.S. at one-, two-, and three-years following diagnosis are 80%, 76%, and 49%, respectively. At present, the only curative therapy is lung transplantation. Alternatively, gene therapy has great promise for producing long-lasting, curative therapeutic effects in pulmonary vascular diseases, including PAH. However, current gene delivery technology must be engineered to improve delivery efficiency to specifically target affected organs, tissue compartments, and cell types, such as the pulmonary vasculature and vascular smooth muscle cells in PAH. Adeno-associated viral (AAV) vectors (distinct from adenoviral vectors) have recently gained significant attention, since unlike most vectors, AAV does not elicit appreciable inflammatory or toxic responses, while providing long-term gene expression in vivo. Despite evidence that recombinant AAV (rAAV) vectors have promise for safe, long-term transgene expression in the lung, a strategy to optimize pulmonary vascular delivery has not been defined, and thus pulmonary vascular-targeted rAAV vectors are not available. Our objectives in this proposal are to identify the AAV serotype (of the currently utilized AAV serotypes, AAV1- 9) best able to transduce the lung vasculature following vascular administration and then to further modify this serotype to enhance lung tropism using a directed evolution approach. We will thereby generate novel AAV serotypes that specifically target the pulmonary vasculature. We hypothesize that engineering AAV serotypes using a directed evolution approach, incorporating selection and amplification in pulmonary endothelial cell culture, will yield novel AAV capsid variants with enhanced tropism to the lung vasculature. We will address this hypothesis in the following aims: Specific Aim 1. To determine the currently known AAV serotype (AAV1-9) with the greatest tropism to pulmonary vascular endothelial cells. Specific Aim 2. To determine whether directed evolution incorporating selection and amplification in cell culture and selection in vivo will yield novel AAV variants with enhanced pulmonary endothelial cell tropism when administered via the vasculature. Completion of these specific aims will result in the development of pulmonary endothelial cell specific vectors. Pulmonary vascular targeting will enable the development and implementation of gene therapeutic modalities as a means of treating pulmonary vascular diseases, including but not limited to pulmonary arterial hypertension. This technology can be generalized to produce AAV vectors with other organ and/or cell type specific tropisms that treat many diseases. Despite recent advances in the medical management of pulmonary arterial hypertension (PAH), survival rates in the U.S. at one-, two-, and three-years following diagnosis are 80%, 76%, and 49%, respectively. Adeno- associated viral vectors have recently gained significant attention. Despite evidence that these vectors have promise for safe, long-term gene therapy use in the lung, strategies need to be developed to move this field forward. Completion of this study will result in the development of lung viral vectors suitable for gene therapy. Furthermore, this technology can be generalized to develop new viral vectors suitable to treat a wide-array of diseases. [unreadable] [unreadable] [unreadable]